U.S. patent application number 13/549988 was filed with the patent office on 2013-01-17 for plume collimation for laser ablation electrospray ionization mass spectrometry.
This patent application is currently assigned to THE GEORGE WASHINTON UNIVERSITY. The applicant listed for this patent is Jessica A. Stolee, Akos Vertes. Invention is credited to Jessica A. Stolee, Akos Vertes.
Application Number | 20130015345 13/549988 |
Document ID | / |
Family ID | 47518407 |
Filed Date | 2013-01-17 |
United States Patent
Application |
20130015345 |
Kind Code |
A1 |
Vertes; Akos ; et
al. |
January 17, 2013 |
Plume Collimation for Laser Ablation Electrospray Ionization Mass
Spectrometry
Abstract
In various embodiments, a device may generally comprise a
capillary having a first end and a second end; a laser to emit
energy at a sample in the capillary to ablate the sample and
generate an ablation plume in the capillary; an electrospray
apparatus to generate an electrospray plume to intercept the
ablation plume to produce ions; and a mass spectrometer having an
ion transfer inlet to capture the ions. The ablation plume may
comprise a collimated ablation plume. The device may comprise a
flow cytometer. Methods of making and using the same are also
described.
Inventors: |
Vertes; Akos; (Reston,
VA) ; Stolee; Jessica A.; (Washington, DC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Vertes; Akos
Stolee; Jessica A. |
Reston
Washington |
VA
DC |
US
US |
|
|
Assignee: |
THE GEORGE WASHINTON
UNIVERSITY
Washington
DC
|
Family ID: |
47518407 |
Appl. No.: |
13/549988 |
Filed: |
July 16, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61507836 |
Jul 14, 2011 |
|
|
|
Current U.S.
Class: |
250/282 ;
250/288 |
Current CPC
Class: |
H01J 49/165 20130101;
H01J 49/0404 20130101; H01J 49/145 20130101; H01J 49/0463 20130101;
H01J 49/167 20130101 |
Class at
Publication: |
250/282 ;
250/288 |
International
Class: |
H01J 49/10 20060101
H01J049/10; H01J 49/26 20060101 H01J049/26 |
Goverment Interests
STATEMENT OF GOVERNMENTAL INTEREST
[0002] This invention was made with Government support under Grant
No. 0719232 awarded by the National Science Foundation and Grant
No. DEFG02-01ER15129 awarded by the U.S. Department of Energy. The
government has certain rights in the invention
Claims
1. A device comprising: a capillary including a first end and a
second end; a pulsed, mid-infrared laser to emit energy at a sample
in the capillary to ablate the sample and generate an ablation
plume in the capillary; an electrospray apparatus to generate an
electrospray plume to intercept the ablation plume exiting the
capillary to produce ions; and a mass spectrometer having an ion
transfer inlet to capture the ions.
2. The device of claim 1, wherein the ablation plume is a
collimated ablation plume.
3. The device of claim 1, wherein the ablation plume is not a
freely expanding ablation plume.
4. The device of claim 1 comprising transmission geometry, wherein
the mid-infrared laser is on a first side of the sample and at
least a portion of the ablation plume is generated on a second side
of the sample.
5. The device of claim 1, wherein the second end of the capillary
comprises an open end and the electrospray apparatus comprises an
electrospray emitter tip, and an angle between the open end of the
capillary and the electrospray emitter tip is about 90.degree..
6. The device of claim 1, wherein the capillary comprises an inner
diameter from 0.1 mm to 5 mm and a length from 1 mm to 5 mm.
7. The device of claim 1, wherein the capillary comprises a
chemically modified interior surface.
8. The device of claim 1 comprising at least one of focusing
optics, an optical fiber, and a hollow waveguide to couple the
energy to the sample in the capillary and deliver the energy to the
sample.
9. The device of claim 8, wherein the optical fiber comprises a
linearly tapered tip.
10. The device of claim 8, wherein a portion of the optical fiber
is positioned inside the capillary and the sample is positioned
inside the capillary intermediate the optical fiber and the second
end of the capillary.
11. The device of claim 8, wherein the capillary comprises a hollow
waveguide.
12. The device of claim 1, wherein the sample comprises water and
the energy is absorbed by the water in the sample, and wherein the
sample is not under vacuum.
13. The device of claim 1, wherein the sample comprises a
suspension of at least one cell in an aqueous solution and a sample
volume from 1 picoliter to 2 microliters.
14. The device of claim 1 comprising a flow cytometer in fluid
communication with the capillary.
15. The device of claim 1 comprising: a flow through capillary to
hydrodynamically focus the sample in a stream of fluid; a
continuous laser on a first side of the flow through capillary to
irradiate the stream of fluid with a focused beam, wherein the
focused beam is upstream from the mid-infrared laser; a detector on
a second side of the flow through capillary to detect when the
sample passes the focused beam; and a delay generator to activate
the mid-infrared laser when the sample is at a point of ablation in
the capillary, wherein the delay generator is in electrical
communication with the detector and the mid-infrared laser.
16. A method comprising: ablating a sample by a mid-infrared laser
pulse in a capillary to generate an ablation plume in the
capillary; intercepting the ablation plume by an electrospray plume
after it exits from the capillary to produce ions; and detecting
the ions by mass spectrometry; wherein the ablation plume is a
collimated ablation plume; and wherein the sample comprises water
and the laser energy is absorbed by the water in the sample.
17. The method of claim 16 comprising collimating the ablation
plume with one of the capillary and a hollow waveguide to generate
the collimated ablation plume.
18. The method of claim 16 comprising ejecting at least a portion
of the ablation plume from the capillary on a side of the sample
opposite from the mid-infrared laser.
19. The method of claim 16 comprising ablating the sample in a
hollow waveguide.
20. The method of claim 16 comprising: hydrodynamically focusing
the sample in a stream of fluid by one of a flow cytometer and a
flow through capillary; irradiating the stream of fluid with a
focused beam from a continuous laser; detecting when the sample
passes the focused beam; and activating the mid-infrared laser when
the sample is at a point of ablation in the capillary to ablate the
sample.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. provisional
application Ser. No. 61/507,836, filed on Jul. 14, 2011, which is
hereby incorporated herein by reference in its entirety.
BACKGROUND
[0003] The apparatuses and methods described herein generally
relate to ionization sources for mass spectrometers and methods of
mass spectrometry, and in particular, laser ablation electrospray
ionization (LAESI) mass spectrometry (MS), as well as methods of
making and using the same.
[0004] Mass spectrometry is an analytical technique that has been
successfully used in chemistry, biology, medicine, and other fields
for qualitative and quantitative analysis. The analysis of a single
cell and/or subcellular component by conventional methods of mass
spectrometry typically requires extensive sample preparation which
may alter the molecular composition of the system. For example,
matrix-assisted laser desorption ionization (MALDI) combined with
laser capture microdissection may suffer from time consuming and
complex sample preparation, e.g., to freeze or fix the sample,
which may cause perturbations to the biological sample. MALDI also
utilizes a matrix that may interfere with the analysis of single
cells and subcellular components. Live video mass spectrometry and
direct organelle mass spectrometry use organic solvents that may
also interfere with the analysis of single cells and subcellular
components. Mass spectrometry may be combined with conventional
separation techniques, such as capillary electrophoresis, however,
these techniques may increase analysis time, complexity and/or
cost.
[0005] Accordingly, more efficient and/or cost-effective mass
spectrometry devices and methods of making and using the same are
desirable.
DESCRIPTION OF THE DRAWINGS
[0006] The various embodiments described herein may be better
understood by considering the following description in conjunction
with the accompanying drawings.
[0007] FIG. 1A includes an image of a freely expanding
hemispherical ablation plume generated by the mid-infrared ablation
of water in the ambient environment.
[0008] FIG. 1B includes a schematic of a collimated ablation plume
according to various embodiments described herein.
[0009] FIGS. 2-6 include illustrations of mass spectrometry systems
according to various embodiments described herein.
[0010] FIG. 7 includes a graph plotting signal intensity and laser
repetition rate (Hz) according to various embodiments described
herein.
[0011] FIG. 8 includes a schematic and geometrical parameters of
radially confined ablation in transmission geometry for plume
collimation according to various embodiments described herein.
[0012] FIGS. 9A-C include illustrations of etched fibers tips for
mass spectrometry systems according to various embodiments
described herein.
[0013] FIG. 10A includes an image of a glass capillary inserted
into a water droplet comprising cells according to various
embodiments described herein.
[0014] FIG. 10B includes an image of about fifteen (15) squamous
epithelial cells after being drawn into a hollow glass capillary by
capillary forces according to various embodiments described
herein.
[0015] FIG. 11 includes illustrations of mass spectrometry systems
according to various embodiments described herein.
[0016] FIG. 12 includes a representative LAESI mass spectrum from
about twenty-five (25) squamous epithelial cells according to
various embodiments described herein. The inset in FIG. 12 includes
an image of about twenty-five (25) stained squamous epithelial
cells. The scale bar in the inset is 50 micrometers.
[0017] FIGS. 13A-E include representative LAESI mass spectra of
bradykinin solution in capillaries having an inner diameter of 2 mm
and a length of 2 mm, 3.8 mm, 5 mm, 6 mm, and 7.7 mm, respectively,
according to various embodiments described herein.
[0018] FIG. 14A includes a representative LAESI mass spectrum of
2.5 .mu.L of 0.1 mM bradykinin solution comprising 50% (v/v) water
and 50% (v/v) methanol in a capillary having an inner diameter of 1
mm and a length of 2.5 mm according to various embodiments
described herein.
[0019] FIG. 14B includes a representative LAESI mass spectrum of 5
.mu.L of 0.1 mM bradykinin solution comprising 50% (v/v) water and
50% (v/v) methanol in a capillary having an inner diameter of 2 mm
and a length of 2 mm according to various embodiments described
herein.
[0020] FIGS. 15A-D include representative LAESI mass spectra of
squamous epithelial cells suspended in a droplet of water according
to various embodiments described herein. FIG. 15A includes a
representative mass spectrum of 20 squamous epithelial cells. FIG.
15B includes a representative mass spectrum of 10 squamous
epithelial cells. FIG. 15C includes a representative mass spectrum
of 6 squamous epithelial cells. FIG. 15D includes a representative
mass spectrum of 4 squamous epithelial cells.
[0021] FIG. 16 includes representative LAESI mass spectrum of about
less than 500 epithelial beta cells having a size of about 5-10
.mu.m suspended in a 2.5 .mu.L droplet of water in a capillary
according to various embodiments described herein. The inset in
FIG. 16 includes an image of a small cell population of about 550
epithelial beta cells prior to ablation.
[0022] FIG. 17 includes a graph plotting signal intensity and
concentration (M) for mass spectrometry systems according to
various embodiments described herein and a mass spectrometry system
lacking a collimated ablation plume. The inset in FIG. 17 includes
representative LAESI mass spectrum of 0.5 .mu.L of
1.2.times.10.sup.-9M verapamil solution comprising 50% (v/v) water
and 50% (v/v) methanol.
DESCRIPTION OF CERTAIN EMBODIMENTS
[0023] As generally used herein, the articles "one", "a", "an" and
"the" refer to "at least one" or "one or more", unless otherwise
indicated.
[0024] As generally used herein, the terms "including" and "having"
mean "comprising".
[0025] As generally used herein, the term "about" refers to an
acceptable degree of error for the quantity measured, given the
nature or precision of the measurements. Typical exemplary degrees
of error may be within 20%, 10%, or 5% of a given value or range of
values. Alternatively, and particularly in biological systems, the
terms "about" refers to values within an order of magnitude,
potentially within 5-fold or 2-fold of a given value.
[0026] All numerical quantities stated herein are approximate
unless stated otherwise. Accordingly, the term "about" may be
inferred when not expressly stated. The numerical quantities
disclosed herein are to be understood as not being strictly limited
to the exact numerical values recited. Instead, unless stated
otherwise, each numerical value is intended to mean both the
recited value and a functionally equivalent range surrounding that
value. At the very least, and not as an attempt to limit the
application of the doctrine of equivalents to the scope of the
claims, each numerical parameter should at least be construed in
light of the number of reported significant digits and by applying
ordinary rounding techniques. Notwithstanding the approximations of
numerical quantities stated herein, the numerical quantities
described in specific examples of actual measured values are
reported as precisely as possible.
[0027] Any numerical range recited in this specification is
intended to include all sub-ranges of the same numerical precision
subsumed within the recited range. For example, a range of "1.0 to
10.0" is intended to include all sub-ranges between (and including)
the recited minimum value of 1.0 and the recited maximum value of
10.0, that is, having a minimum value equal to or greater than 1.0
and a maximum value equal to or less than 10.0, such as, for
example, 2.4 to 7.6. Any maximum numerical limitation recited in
this disclosure is intended to include all lower numerical
limitations subsumed therein and any minimum numerical limitation
recited in this disclosure is intended to include all higher
numerical limitations subsumed therein. Accordingly, Applicants
reserve the right to amend this specification, including the
claims, to expressly recite any sub-range subsumed within the
ranges expressly recited herein.
[0028] In the following description, certain details are set forth
in order to provide a better understanding of various embodiments
of ionization sources for mass spectrometers and methods for making
and using the same. However, one skilled in the art will understand
that these embodiments may be practiced without these details
and/or in the absence of any details not described herein. In other
instances, well-known structures, methods, and/or techniques
associated with methods of practicing the various embodiments may
not be shown or described in detail to avoid unnecessarily
obscuring descriptions of other details of the various
embodiments.
[0029] This disclosure describes various features, aspects, and
advantages of various embodiments of ionization sources for mass
spectrometers and methods for making and using the same. It is
understood, however, that this disclosure embraces numerous
alternative embodiments that may be accomplished by combining any
of the various features, aspects, and advantages of the various
embodiments described herein in any combination or sub-combination
that one of ordinary skill in the art may find useful. Such
combinations or sub-combinations are intended to be included within
the scope of this specification. As such, the claims may be amended
to recite any features or aspects expressly or inherently described
in, or otherwise expressly or inherently supported by, the present
disclosure. Further, Applicants reserve the right to amend the
claims to affirmatively disclaim any features or aspects that may
be present in the prior art. The various embodiments disclosed and
described in this disclosure may comprise, consist of, or consist
essentially of the features and aspects as variously described
herein.
[0030] According to certain embodiments, more efficient and/or
cost-effective mass spectrometry devices and methods of making and
using the same are described.
[0031] Metabolism generally refers to chemical processes of a
living cell or organism that support and maintain life. The
products of these chemical processes may be generally referred to
as metabolites. The metabolites and distribution of metabolites in
a cell or tissue may change depending on its function, biological
state, developmental stage, history, and/or environment.
Identification and analysis of metabolites and metabolite
distributions may facilitate a better understanding of cell
function. Certain embodiments may be used to analyze cellular
heterogeneity and provide insight into the cell-to-cell variations
of metabolic pathways affected by diseases.
[0032] Mass spectrometric analysis of subcellular components,
single cells, and/or groups of cells may be limited by small sample
volumes and/or inefficient ion production. A small sample volume
may coexist with a low concentration of subcellular components in a
single cell or group of cells. Some conventional techniques to
isolate single cells and/or groups of cells may cause
sampling-related perturbations that disrupt metabolite
distributions within the sample. Therefore, mass spectrometry
devices and methods of using the same having improved ion
efficiency and/or sensitivity and/or limits of detection are
desirable.
[0033] Some mass spectrometry techniques may comprise a freely
expanding ablation plume, such as a hemispherical laser ablation
plume illustrated in FIG. 1A, characterized by low ionization
efficiency and/or low sensitivity and/or low limits of detection. A
freely expanding ablation plume in the ambient environment may be
generated, for example, when mid-infrared laser pulses at a
wavelength of about 2.94 .mu.m and a pulse length of about 5
nanoseconds are emitted at a sample comprising water. The
absorption of the laser energy by the water may initiate surface
evaporation, shock-wave emission, and/or ejection of droplets via
phase explosion. The surface evaporation may initiate relatively
slow plume expansion that, after about 300 ns, induces a
propagation of shock waves, which may not be very efficient.
However, at the spinodal limit, i.e., when the sample is
superheated to about its critical temperature, a rapidly expanding
vapor plume, droplets, and/or particulates may be ejected. Without
wishing to be bound to any particular theory, it is believed that
the spinodal limit may be achieved when the volumetric energy
density, .epsilon.(.epsilon.=.mu.F, where F is the laser fluence
and .mu. is the absorption coefficient), is greater than about 1
kJ/cm.sup.3 or when the laser energy increases the temperature of
the liquid to about 80% of its critical temperature. After about 1
.mu.s, the phase explosion may induce recoil pressure that
generates an efficient secondary material ejection process.
[0034] According to certain embodiments, mass spectrometry devices
and methods of making and using the same may be characterized by
improved ionization efficiency and/or improved sensitivity and/or
improved limits of detection relative to a freely expanding
ablation plume. As described herein, laser ablation may be used to
eject a small volume from a sample in a collimated ablation plume
to improve ion production, and thereby ionization efficiency and/or
limits of detection. In various embodiments, mass spectrometry
devices and methods of making and using the same may comprise
direct mass spectrometry devices and methods of making and using
the same for in vivo analysis of small cell populations, single
cells and/or subcellular components. In various embodiments, a
biological sample may be analyzed in a native environment with
minimal and/or no sample preparation.
[0035] In various embodiments, mass spectrometry devices may
comprise a capillary or hollow waveguide to select a sample for
ablation and/or collimate the ablation plume. For example, a
capillary may be inserted into an aqueous droplet comprising cells
to select one or more cells for ablation. The cell may be drawn
into the capillary by capillary forces. The mass spectrometry
device may comprise ablation in transmission geometry. As shown in
FIG. 1B, a capillary may collimate an ablation plume generated in
the capillary. In various embodiments, the ablation plume may
comprise a collimated ablation plume. The collimated ablation plume
may comprise a radially confined ablation plume. The collimated
ablation plume may comprise a collinear ablation plume. The
capillary may reduce and/or eliminate the radial expansion of the
ablation plume. The collimated ablation plume may be ejected from
the capillary. The collimated ablation plume may generate a more
efficient ionization process.
[0036] A collimated ablation plume in the ambient environment may
be generated, for example, when mid-infrared laser pulses at a
wavelength of about 2.94 .mu.m and a pulse length of about 5
nanoseconds are emitted at a sample comprising water within a
capillary. The radial expansion of the ablation plume may be
reduced and/or eliminated by the capillary. Without wishing to be
bound to any particular theory, the collimated ablation plume may
exhibit different photomechanical effects, plume dynamics, and/or
kinetics relative to the freely expanding ablation plume. For
example, the collimated ablation plume may achieve greater
pressures and/or greater temperatures than the freely expanding
ablation plume. Further, when an optical fiber is used to couple
the laser energy to the sample, the optical fiber tip may generate
acoustic radiation that causes greater tensile stress in the water,
and may generate explosive vaporization of the water, cavitation of
the water, and/or bubble formation. The collapse of the generated
bubble may generate the ejection of the ablation plume in a high
speed liquid jet. The capillary may generate more efficient plume
collimation and acceleration. The radial confinement of the
ablation plume in the capillary may generate increased pressures in
the capillary, resulting in forward directed propulsion of a
collimated ablation plume. The collimated ablation plume may
improve ion formation and/or ion efficiency.
[0037] Certain embodiments of the LAESI ionization sources for mass
spectrometers and methods of making and using the same described
herein may provide certain advantages over other approaches of mass
spectrometric analysis. The advantages may include one or more of,
but are not limited to, in situ analysis, in situ single cell
analysis, in situ subcellular analysis, in vivo analysis, in vivo
single cell analysis, in vivo subcellular analysis, simultaneous
detection of multiple components in samples, independent
optimization of ablation conditions and ionization conditions, a
wider dynamic range of samples that may be used, quantitative
analysis, semi-quantitative analysis, operation under ambient
conditions, simpler sample preparation, minimal sample
manipulation, minimal sample degradation, direct analysis of
tissues and cells, analysis of large samples, two-dimensional mass
spectrometric imaging at atmospheric pressure, three- dimensional
mass spectrometric imaging at atmospheric pressure, the ability to
monitor environmental effects or external stimuli on multiple
cells, single cells, or subcellular components, the ability to
monitor the effects of xenobiotics, for example, pharmaceuticals,
drug candidates, toxins, environmental pollutants, and/or
nanoparticles, the ability to couple with a flow cytometry system,
higher throughput, improved sampling time, positional sensitivity,
improved sensitivity, improved sensitivity to surface properties,
improved ionization, improved ionization efficiency, and improved
detection limits.
[0038] Laser ablation electrospray ionization mass spectrometry may
be generally described in the following U.S. Patents and U.S.
Patent Applications: U.S. Pat. No. 7,964,843, entitled
"Three-dimensional molecular imaging by infrared laser ablation
electrospray ionization mass spectrometry", which issued on Jun.
21, 2011; U.S. Pat. No. 8,067,730, entitled "Laser Ablation
Electrospray Ionization (LAESI) for Atmospheric Pressure, In Vivo,
and Imaging Mass Spectrometry", which issued on Nov. 29, 2011; and
U.S. Patent Application Publication No. 2010/0285446 entitled
"Methods for Detecting Metabolic States by Laser Ablation
Electrospray Ionization Mass Spectrometry", which was filed on May
11, 2010.
[0039] In various embodiments, a device may generally comprise a
capillary having a first end and a second end, a laser system to
emit energy at a sample in the capillary to ablate the sample and
generate an ablation plume in the capillary, an electrospray
apparatus to generate an electrospray plume to intercept the
ablation plume to produce ions, and a mass spectrometer system. At
least one of the first end and second end may comprise an open end.
In certain embodiments, the first end may comprise an open end and
the second end may comprise an open end. In certain embodiments,
the first end may comprise a closed end and the second end may
comprise an open end. The mass spectrometer system may comprise a
mass spectrometer having an ion transfer inlet to capture the ions,
and a recording device, such as, for example, a personal computer.
The electrospray plume may intercept the ablation plume when the
ablation plume exits the second end of the capillary. The ablation
plume may comprise a collimated ablation plume, such as, for
example, a radially confined ablation plume and/or a collinear
ablation plume. In certain embodiments, the capillary may comprise
a glass capillary. In certain embodiments, the capillary may
comprise a hollow waveguide.
[0040] The laser system may comprise a mid-infrared laser and a
focusing system comprising fiber optics, coupling lenses, focusing
lenses, and/or an optical fiber. The focusing system may deliver
and/or couple the laser pulses to the sample. The electrospray
apparatus may comprise an electrospray ionization emitter having a
power supply and a syringe pump. The device may comprise a sample
mount. The device may comprise a shroud to enclose the sample, the
sample mount, and/or the electrospray emitter. The sample
environment may be temperature controlled and/or atmosphere
controlled. The atmosphere may comprise ambient pressure and
temperature. The pressure may range from 0.1-5 atm, such as, for
example, 0.5-5 atm, 1-5 atm, and 0.1-1 atm. The temperature may
range from -10.degree. C. to 60.degree. C. The relative humidity
may range from 10% to 90%.
[0041] In various embodiments, a device may comprise a capillary
having a first end and a second end, a pulsed, mid-infrared laser
to emit energy at a sample in the capillary to ablate the sample
and generate an ablation plume in the capillary, an electrospray
apparatus to generate an electrospray plume to intercept the
ablation plume to produce positive or negative ions, and a mass
spectrometer having an ion transfer inlet to capture the ions.
Referring to FIG. 2, a device may comprise a laser 1, such as, for
example, a pulsed, mid-infrared laser, a focusing device, e.g., a
lens (not shown), a fiber mount 2, an optical fiber 3, a sample ( )
contained in the capillary 4, an electrospray apparatus comprising
an emitter 9, a high voltage power supply 10, a syringe pump 11,
and a mass spectrometer 12. The laser may be one of an Er:YAG
laser, a Nd:YAG laser driven optical parametric oscillator and a
free electron laser. The capillary 4 may comprise at least a
portion of the sample. The sample may be positioned intermediate a
first end of the capillary 4 and a second end of the capillary 4.
The sample may be positioned adjacent or proximate to the open end
of the capillary 4. At least a portion of the sample may be
positioned outside the capillary. The sample may be positioned
intermediate the optical fiber 3 and the second end of the
capillary 4. The laser 1 may be coupled to the first end of the
capillary 4. The laser pulse may be delivered and/or coupled to the
sample by the optical fiber 3. The device may comprise one or more
actuators (not shown) to position the focusing device, capillary,
electrospray emitter, and/or laser. The device may comprise a
recording device (not shown).
[0042] In various embodiments, the electrospray plume () may
intercept the ablation plume ( ) to generation ions (+) detectable
by the mass spectrometer 12. Depending on the polarity of the
electrospray, the ions may be positive or negative. In at least one
embodiments, the ions may comprise cations. As shown in FIG. 2, the
electrospray plume () may travel in a forward direction from the
emitter 9 toward the orifice of the mass spectrometer 12. The
capillary 4 may be oriented toward the electrospray plume (). The
second end of the capillary may be oriented towards the
electrospray plume () At least a portion of the ablation plume ( )
may be generated in the capillary 4. The ablation plume ( ) may be
generated in the capillary 4. The ablation plume ( ) may travel in
a forward direction toward the second end of the capillary 4. The
capillary 4 may radially confine the ablation plume ( ). The
ablation plume ( ) may comprise a collimated ablation plume. The
collimated ablation plume may comprise a radially confined ablation
plume. The collimated ablation plume may comprise a collinear
ablation plume. At least a portion of the ablation plume ( ) may be
ejected from the capillary 4. The ablation plume ( ) may be ejected
from the capillary 4. The ablation plume ( ) may be ejected from
the second end of the capillary towards the electrospray plume ()
The ejected ablation plume may be a collimated ablation plume. The
electrospray plume () may intercept the ablation plume ( ) to
produce ions detectable by the mass spectrometer 12. Without
wishing to be bound to any particular theory, the collimated
ablation plume may improve ion formation and/or ionization
efficiency.
[0043] Referring to FIGS. 3 and 4, according to certain
embodiments, the mass spectrometer 12 orifice may be on one of a
same axis or a different axis as the electrospray emitter 9. The
x-y-z axes may be orientated with respect to the mass spectrometer
12. The x'-y'-z' axes may be orientated with respect to the
electrospray emitter 9. The x'-y'-z' axes may be parallel to the
x-y-z axes, respectively. The x''-y''-z'' axes may be parallel to
the x-y-z axes, respectively. As shown in the FIG. 3, the mass
spectrometer 12, electrospray emitter 9, and the second end of the
capillary 4 may be in the same x-y plane. The distance 15 from the
mass spectrometer 12 orifice to the electrospray emitter 9 tip
along the x-axis may be from 1 mm to 20 mm, such as, for example, 5
mm to 15 mm, 5 mm, 10 mm, and 15 mm. In at least one embodiment,
the distance 15 may be 12 mm. The distance 16 from the mass
spectrometer 12 orifice to the electrospray emitter 9 tip along the
y-axis may be from -20 mm to 20 mm, such as, for example, -10 mm,
-5 mm, -1 mm, 0 mm, 1 mm, 5 mm, and 10 mm. The distance 21 from the
mass spectrometer 12 orifice to the electrospray emitter 9 tip
along the z-axis may be from -20 mm to 20 mm, such as, for example,
-10 mm, -5 mm, -1 mm, 0 mm, 1 mm, 5 mm, and 10 mm. The angle 18 may
be defined as the angle between the central axis of the mass
spectrometer 12 orifice along the x-axis and the axis of the
electrospray emitter 9, or more generally, as the angle between the
axis of the electrospray emitter 9 and the x'-z' plane illustrated
in FIG. 4. The angle 20 may be defined as the angle between the
projection of the electrospray emitter 9 axis to the x'-z' plane
and the x'-axis. Each of the angles 18 and 20 may be individually
selected from -90.degree. to 90.degree., such as, for example,
-45.degree.to 45.degree., -60.degree., -45.degree., -30.degree.,
-15.degree. , 0.degree., 15.degree., 30.degree., 45.degree.,
60.degree., and 90.degree..
[0044] Referring to FIGS. 3 and 4, according to certain
embodiments, the distance 13 from the front of the mass
spectrometer 12 orifice to the second end of the capillary 4 along
the x-axis (the y-z plane illustrated in FIG. 4) may be 0-20 mm,
such as, for example, 1 mm, 5 mm, 10 mm, and 15 mm. The distance 14
from the central axis of the mass spectrometer 12 orifice to the
second end of the capillary 4 along the y-axis (the x-z plane
illustrated in FIG. 4) may be from -20 mm to 20 mm, such as, for
example, -10 mm, -1 mm, 0 mm, 1 mm, and 10 mm. The distance 22 from
the mass spectrometer 12 orifice to the second end of the capillary
4 along the z-axis (the x-y plane illustrated in FIG. 4) may be
from -20 mm to 20 mm, such as, for example, -10 mm, -1 mm, 0 mm, 1
mm, and 10 mm. The angle 17 may be defined as the angle between the
projection of the capillary 4 axis to the x''-z'' plane and the
z''-axis illustrated in FIG. 4. The angle 19 may be defined as the
angle between the axis of the capillary 4 and the x''-z'' plane
illustrated in FIG. 4. Each of the angles 17 and 19 may be
individually selected from -90.degree. to 90.degree., such as, for
example, -45.degree. to 45.degree., -90.degree., -60.degree.,
-45.degree., -30.degree., 0.degree., 30.degree., 45.degree.,
60.degree., and 90.degree.. In various embodiments, the second end
of the capillary 4 may be 15 mm above or below the x-y plane. In at
least one embodiment, the electrospray solution may be applied on
axis with the mass spectrometer 12 orifice (angles 20 and
18=0.degree. and distances 21 and 16=0 mm) In at least one
embodiment, the electrospray solution may be applied at a right
angle)(90.degree.) into the ablation plume.
[0045] In various embodiments, the distance 13 may be from 0 mm to
20 mm, such as, for example greater than 0 mm to 20 mm, and 4.5 mm,
the distance 14 may be from -20 mm to 20 mm, such as, for example,
-10 mm, the distance 15 may be from greater than 0 mm to 20 mm,
such as, for example, 1 and 12 mm, the distance 16 may be from -20
mm to 20 mm, such as, for example, 0 mm, the distance 21 may be
from -20 mm to 20 mm, such as, for example, 0 mm, and the distance
22 may be from -20 mm to 20 mm, such as, for example, 0 mm, and the
angle 17 may be from -90.degree. to 90.degree., such as, for
example, 0.degree., the angle 18 may be from -90.degree. to
90.degree., such as, for example, 0.degree., the angle 19 may be
from -90.degree. to 90.degree., such as, for example, 0.degree.,
and the angle 20 may be from -90.degree. to 90.degree., such as,
for example, 0.degree..
[0046] In various embodiments, a device may generally comprise a
flow cytometer. In various embodiments, a device may comprise a
flow cytometry system comprising a capillary, a laser system to
emit energy at a sample in the capillary to ablate the sample and
generate an ablation plume in the capillary, an electrospray
apparatus to generate an electrospray plume to intercept the
ablation plume to produce ions, and a mass spectrometer system. The
flow cytometry system may comprise a flow cytometer. The flow
cytometry system may comprise a flow through capillary having an
open end and an opposite end, and optionally, a waste container
positioned adjacent the open end of the capillary. The opposite end
of the capillary may comprise a closed end. The mass spectrometer
system may comprise a mass spectrometer having an ion transfer
inlet to capture the ions, and a recording device, such as, for
example, a personal computer. The laser system may comprise a
mid-infrared laser and a focusing system comprising fiber optics,
coupling lenses, and/or focusing lenses. The device may comprise an
optical fiber to deliver and/or couple the laser pulses to the
sample. The electrospray apparatus may comprise an electrospray
ionization emitter having a power supply and a syringe pump. The
device may comprise a sample mount. The device may comprise a
shroud to enclose the sample, the sample mount, and/or the
electrospray emitter.
[0047] The flow cytometry system may hydrodynamically focus a
sample in a stream of fluid. For example, the flow through
capillary may hydrodynamically focus a group of cells into a single
stream of cells. The device may comprise a flow cytometer to
hydrodynamically focus a sample in a stream of fluid. The device
may comprise a focusing system to deliver and/or couple the laser
pulse to the sample when the sample is at a point of ablation in
the capillary. The ablation plume may travel in a forward direction
toward the open end of the capillary. The capillary may radially
confine the ablation plume. The ablation plume may comprise a
collimated ablation plume. The capillary may be oriented toward the
electrospray plume. The ablation plume may be ejected from the
capillary toward the electrospray plume. The ablation plume may be
intercepted by an electrospray plume and ionized to generate ions
detectable by the mass spectrometer.
[0048] In various embodiments, the flow cytometry system may
comprise a continuous laser, such as, for example, an argon ion
laser and a helium-neon (HeNe) laser, positioned on a first side of
the flow through capillary, and a detector, such as, for example, a
photodetector and a fluorescence detector, positioned on a second
side of the flow through capillary, and a delay generator in
electrical communication with the detector and mid-infrared laser.
The continuous laser may be positioned upstream from the
mid-infrared laser. The continuous laser may irradiate the flow
through capillary with a continuous laser beam. The continuous
laser beam may be deflected or scattered by the sample when the
sample passes the continuous laser beam. The detector may detect
the deflected or scattered laser beam and activate the delay
generator. The delay generator may activate the mid-infrared laser
when the sample is at a point of ablation in the capillary. The
delay generator may be configured to delay activation of the
mid-infrared laser until the sample is at a point of ablation in
the capillary. The duration of the delay may be the time for the
sample to travel from the point when the cell intercepts the
continuous laser beam to the point of ablation. In various
embodiments, the sample may comprise a fluorescent tag, such as,
for example, a green fluorescent protein, a yellow fluorescent
protein, an immunofluorescent tag, and an acridine orange dye.
[0049] Referring to FIG. 5, in certain embodiments, a mass
spectrometer device may comprise a mid-infrared laser 1, such as,
for example, a Nd:YAG laser driven optical parametric oscillator, a
focusing system comprising an optical fiber 3 held on one end by a
fiber mount 2, a waste container 4, a capillary 5, a continuous
laser 6, a detector 7, a delay generator 8 in electrical
communication with the detector 7 and mid-infrared laser 1, an
electrospray apparatus including an electrospray emitter 9, a
syringe pump 11, a high voltage power supply 10, and a mass
spectrometer 12. The focusing system may focus the laser pulse
inside the capillary 5 to deliver the laser energy to the sample (
). The device may comprise one or more actuators (not shown) to
position the focusing system, capillary, electrospray emitter,
and/or lasers. The device may comprise a recording device (not
shown).
[0050] Referring to FIG. 6, in certain embodiments, a mass
spectrometer device may comprise a mid-infrared laser 1, such as,
for example, a Nd:YAG laser driven optical parametric oscillator, a
focusing system comprising a beam steering device 21, such as, for
example, a mirror, and a focusing device 22, such as, for example,
a lens, a waste container 4, a capillary 5, a continuous laser 6, a
detector 7, a delay generator 8 in electrical communication with
the detector 7 and mid-infrared laser 1, an electrospray apparatus
including an electrospray emitter 9, a syringe pump 11, a high
voltage power supply 10, and a mass spectrometer 12. The focusing
system may focus the laser pulse inside the capillary 5 to deliver
the laser energy to the sample ( ). The device may comprise one or
more actuators (not shown) to position the focusing system,
capillary, electrospray emitter, and/or lasers. The device may
comprise a recording device (not shown).
[0051] Regarding FIGS. 5 and 6, the point of ablation may be
intermediate the open end of the capillary 5 and the point when the
sample passes the continuous laser beam. The point of ablation may
be directly adjacent to the open end of the capillary 5. The
ablation plume may be generated in the capillary 5. The ablation
plume may travel in a forward direction toward the open end of the
capillary 5. The capillary 5 may radially confine the ablation
plume. The ablation plume may comprise a collimated ablation plume.
The capillary 5 may be oriented toward the electrospray plume. The
ablation plume may be ejected from the capillary 5 toward the
electrospray plume. The ablation plume may be intercepted by an
electrospray plume and ionized to generate ions detectable by the
mass spectrometer 12.
[0052] In various embodiments, the laser pulse may have a
wavelength of 100 nm to 8 .mu.m, a diameter of 0.5-20 mm before
focusing, a pulse length of less than one picosecond to 100 ns, and
a repetition rate of up to 100 MHz, such as, for example, 0.1 Hz to
100 MHz, under ambient conditions. In various embodiments, the
laser pulse may have a wavelength of 100 nm to 400 nm, such as 300
nm. In various embodiments, the laser pulse may have a wavelength
of 700 nm to 3000 nm and 2000 nm to 4000 nm , such as, for example,
800 nm and 2940 nm . In various embodiments, the laser pulse may
have a wavelength of 2 .mu.m to 4 .mu.m, such as, for example,
about 3 .mu.m. In various embodiments, the laser pulse may have a
diameter of 0.5 mm to 1 mm, 1 mm to 20 mm, and 1 mm to 5 mm before
focusing. In various embodiments, the laser pulse may have a pulse
length of 200 fs to 10 ns, 1 ns to 100 ns, and 1 ns to 5 ns. In
various embodiments, the laser pulse may have a repetition rate up
to 100 Hz, such as, for example, 0.1 Hz to 100 Hz. In at least one
embodiment, the laser pulse may have a wavelength of 800 nm , a
diameter of 1 mm, and a pulse length of 200 fs. In at least one
embodiment, the laser pulse may have a wavelength of 100 nm to 400
nm , a diameter of 1 mm to 5 mm, and a pulse length of 1 ns to 100
ns. In at least one embodiment, the laser pulse may have a
wavelength of 2940 nm , a diameter of 1 to 20 mm, and a pulse
length of 5 ns. In at least one embodiment, the laser may comprise
a mid-infrared pulsed laser operating at a wavelength from 2600 nm
to 3450 nm , a diameter of 1 to 20 mm, a pulse length from 0.5 ns
to 50 ns, and a repetition rate from 1 Hz to 100 Hz. The energy of
a laser pulse before coupling into the optical fiber may be from
0.1 mJ to 6 mJ, and the pulse-to-pulse energy stability generally
corresponds to 2% to 10%. In at least one embodiment, the energy of
a laser pulse before coupling into the optical fiber may be
554.+-.26 .mu.J, thus the pulse-to-pulse energy stability
corresponds to 5%. The laser system may be operated at 100 Hz for a
period from 0.01 seconds to 20 seconds to ablate a sample. In at
least one embodiment, laser system may be operated at 100 Hz for a
period of 1 second to ablate a sample. In certain embodiments, 1 to
100 laser pulses may be delivered to ablate a sample.
[0053] In various embodiments, the signal intensity may relate to
the repetition rate of the laser pulse. Without wishing to be bound
to any particular theory, the repetition rate may affect the
ablation plume kinetics and/or ablation plume dynamics during plume
collimation. The signal intensity and repetition rate may relate to
laser, the laser pulse, the dimensions of the optical fiber, the
dimensions of the capillary, and/or sample volume. For example,
FIG. 7 shows a graph plotting the signal intensity and laser
repetition rate (Hz) for a 1.5 .mu.L of 1.times.10.sup.-4M
verapamil solution comprising 50% (v/v) water and 50% (v/v)
methanol in a capillary including an inner diameter of 1 mm and a
length of 4.2 mm. As shown in FIG. 7, a repetition rate of about 25
Hz may generate the highest signal intensity. In various
embodiments, the laser pulse may have a repetition rate from 1 Hz
to 100 Hz, such as, for example, up to 50 Hz, 0.1-50 Hz, 5-50 Hz,
15-35 Hz, 20-30 Hz, 20-25 Hz, 25-30 Hz, 22-28 Hz, 23-27 Hz, and 25
Hz. In at least one embodiment, the laser pulse may have a
repetition rate from 20 Hz to 30 Hz. In at least one embodiment,
the laser pulse may have a repetition rate of 25 Hz.
[0054] In various embodiments, the laser may be selected from the
group consisting of a UV laser, a laser emitting visible radiation,
and an infrared laser, such as, for example, a mid-infrared laser.
The UV laser may include, but is not limited to, an excimer laser,
a frequency tripled Nd:YAG laser, a frequency quadrupled Nd:YAG
laser, and a dye laser. The laser emitting visible radiation may
include, but is not limited to, a frequency doubled Nd:YAG laser,
and a dye laser. The infrared laser may include, but is not limited
to, a carbon dioxide laser, a Nd:YAG laser, and a titanium-sapphire
laser. The laser may comprise a tunable titanium-sapphire
mode-locked laser to generate laser pulses having a 800 nm
wavelength, a 1 mm diameter, 200 fs pulse length, 76 MHz repetition
rate, and 5 nJ energy per pulse. The laser system may comprise a
tunable titanium-sapphire mode-locked laser and a regenerative
amplifier associated with the titanium-sapphire laser to generate
laser pulses having a 800 nm wavelength, 200 fs pulse length, 1 kHz
repetition rate, and 1 mJ energy per pulse. A tunable
titanium-sapphire mode-locked laser is commercially available from
Coherent (Santa Clara, Calif.) under the trade designation Mira
900. A regenerative amplifier is commercially available from
Positive Light (Los Gatos, Calif.) under the trade designation
Spitfire.
[0055] In various embodiments, the mid-infrared laser may comprise
one of an Er:YAG laser and a Nd:YAG laser driven optical parametric
oscillator (OPO). The mid-infrared laser may operate at a
wavelength from 2600 nm to 3450 nm , such as 2800 nm to 3200 nm ,
and 2930 nm to 2950 nm. The laser may comprise a mid-infrared
pulsed laser operating at a wavelength from 2600 nm to 3450 nm, in
a pulse on demand mode, or with a repetition rate from 1 Hz to 5000
Hz, and a pulse length from 0.5 ns to 100 ns. In various
embodiments, the laser pulse may have a wavelength at an absorption
band of an OH group. In various embodiments, the mid-infrared laser
may comprise a diode pumped Nd:YAG laser-driven optical parametric
oscillator (OPO) (Vibrant IR, Opotek, Carlsbad, Calif.) operating
at 2940 nm, 100 Hz repetition rate, and 5 ns pulse length.
[0056] In various embodiments, the focusing system may comprise one
or more mirrors, one or more coupling lenses, and/or an optical
fiber. The laser pulse may be steered by gold-coated mirrors
(PF10-03-M01, Thorlabs, Newton, N.J.) and coupled into the cleaved
end of the optical fiber by a plano-convex calcium fluoride lens
(Infrared Optical Products, Farmingdale, N.Y.) having a focal
length from 1 mm to 100 mm, such as 25 mm to 75 mm, and 40 mm to 60
mm. In at least one embodiment, the focal length of the coupling
lens may be 50 mm. In certain embodiments, the optical fiber may
comprise at least one of a GeO.sub.2-based glass fiber, a fluoride
glass fiber, and a chalcogenide fiber. In various embodiments, the
optical fiber may comprise a germanium oxide (GeO.sub.2)-based
glass optical fiber (450 .mu.m core diameter, HP Fiber, Infrared
Fiber Systems, Inc., Silver Spring, Md.) and the laser pulse may be
coupled into the optical fiber by a plano-convex CaF.sub.2 lens
(Infrared Optical Products, Farmingdale, N.Y.). A high-performance
optical shutter (SR470, Stanford Reseach Systems, Inc., Sunnyvale,
Calif.) may be used to select the laser pulses. One end of the
optical fiber may be held by a bare fiber chuck (BFC300, Siskiyou
Corporation, Grants Pass, Oreg.) attached to a five-axis translator
(BFT-5, Siskiyou Corporation, Grants Pass, Oreg.) or a
micromanipulator (MN-151, Narishige, Tokyo, Japan) to adjust the
distance between the fiber tip and the sample.
[0057] In various embodiments, the device may comprise a
visualization system. In various embodiments, the visualization
system may comprise a video microscope system. In case of
transparent sample capillaries, the distance between the fiber tip
and sample surface may be monitored by a long distance video
microscope positioned orthogonal to the capillary (InFocus Model
KC, Infinity, Boulder Colo.) with a 5.times. infinity corrected
objective lens (M Plan Apo 5.times., Mitutoyo Co., Kanagawa,
Japan), and the image may be captured by a CCD camera (Marlin F131,
Allied Vision Technologies, Stadtroda, Germany). When the
environmental vibration is in the low micrometer range, an
approximate distance from 30 .mu.m to 40 .mu.m may be maintained
between the fiber tip and the sample. A similar video microscope
system may be positioned on axis with the capillary to align the
fiber tip within the capillary over the location of interest in the
sample for ablation. The visualization system may comprise a
7.times. precision zoom optic (Edmund Optics, Barrington, N.J.),
fitted with a 5.times. infinity-corrected long working distance
objective lens (M Plan Apo 5.times., Mitutoyo Co., Kanagawa, Japan)
or a 10.times. infinity-corrected long working distance objective
lens (M Plan Apo 10.times., Mitutoyo Co., Kanagawa, Japan) and a
CCD camera (Marlin F131, Allied Vision Technologies, Stadtroda,
Germany). During this alignment, a HeNe laser beam may be coupled
into the optical fiber to highlight the position of the fiber tip.
The HeNe laser beam may replace the mid-IR laser beam during this
alignment.
[0058] In various embodiments, the electrospray apparatus may
comprise a low noise syringe pump 11 (Physio 22, Harvard Apparatus,
Holliston, Mass.) to supply the electrospray solution to a tapered
emitter 9 (inner diameter 50 .mu.m, MT320-50-5-5, New Objective,
Woburn, Mass.) at a constant flow rate. The low noise syringe pump
11 may supply the electrospray solution at a rate from 10 nL/min to
100 .mu.L/min, such as, for example, 200 nL/min and 300 nL/min. The
tapered emitter 9 may have an outside diameter from 100 .mu.m to
500 .mu.m and an inside diameter from 10 .mu.m to 200 .mu.m. The
power supply 10 (PS350, Stanford Research Systems, Sunnyvale,
Calif.) may comprise a regulated power supply to provide a stable
high voltage from 0 to 5 kV to the electrospray emitter, such as,
for example, 2,500 V and 3,100 V. The electrospray solution may
comprise at least one of 50% (v/v) methanol with 0.1% (v/v) acetic
acid, 50% (v/v) methanol with 0.1% (v/v) formic acid, 50% (v/v)
methanol with 0.1% (v/v) trifluoroacetic acid, 50% (v/v) methanol
with 0.1% (w/v) ammonium acetate. In various embodiments, to
generate the electrospray plume, the electrospray solution may
comprise 50% (v/v) aqueous methanol solution with 0.1% (v/v) acetic
acid pumped through the tapered emitter 9 at a flow rate of 300
nL/min by the syringe pump 11 and 3,100 V may be applied by the
power supply 10.
[0059] In certain embodiments, the atmosphere and/or the
electrospray solution may comprise a reactant to facilitate the
ionization and/or fragmentation of certain constituents of the
sample. The electrospray solution may comprise reactants to
facilitate ion formation or to produce ions with desirable
properties (e.g., with enhanced fragmentation properties). For
example, the electrospray solution may comprise Li.sub.2SO.sub.4 to
facilitate the structural identification of lipids by inducing
structure specific fragmentation in collision induced dissociation
experiments. Examples of reactive gases include, but are not
limited to, ammonia, SO.sub.2, and NO.sub.2.
[0060] The ions may be detected and/or analyzed by a mass
spectrometer. The mass spectrometer may comprise an orthogonal
acceleration time-of-flight mass spectrometer (Q-TOF Premier,
Waters Co., Mass.). The orifice of the mass spectrometer may have
an inner diameter from 100 .mu.m to 500 .mu.m, such as, for
example, 225 .mu.m to 375 .mu.m. In at least one embodiment, the
orifice of the mass spectrometer may have an inner diameter from
100 .mu.m to 200 .mu.m, such as, for example, 127 .mu.m. The
orifice of the mass spectrometer may be extended by a straight or
curved extension tube having a similar inner diameter as the
orifice of the mass spectrometer and a length from 20 mm to 500 mm.
The interface block temperature may be from ambient temperature to
150.degree. C., such as, for example, 23.degree. C. to 90.degree.
C. and 60.degree. C. to 80.degree. C. In at least one embodiment,
the interface block temperature may be 80.degree. C. The potential
may be from -100 V to 100 V, such as, for example, -70 V to 70 V.
In at least one embodiment, the potential may be -70 V. Tandem mass
spectra may be obtained by collision activated dissociation (CAD)
with a collision gas, such as argon, helium or nitrogen, at a
collision cell pressure from 10.sup.-6 mbar to 10.sup.-2 mbar, and
with collision energies from 10 eV to 200 eV. In at least one
embodiment, the collision gas may be argon, the collision cell
pressure may be 4.times.10.sup.-3 mbar, and the collision energies
may be from 10 eV to 25 eV.
[0061] In various embodiments, the device may comprise one of
transmission geometry and reflection geometry. In reflection
geometry, the laser and ablation plume may be on the same side of
the sample. For example, the laser may be positioned on one side of
the sample and the ablation plume may be generated on the same
side. In transmission geometry, the laser may be positioned on a
first side of the sample and the ablation plume may be generated on
a second side of the sample. For example, the laser may emit energy
at the rear of the sample to generate an ablation plume on the
front of the sample. In transmission geometry, at least a portion
of the ablation plume or at least a substantial portion of the
ablation plume may be on a side opposite from the laser, and at
least a portion of the ablation plume or no portion of the ablation
plume may be on the same side as the laser.
[0062] In transmission geometry, the ablation plume may be
generated in the capillary. The ablation plume may travel in a
forward direction away from the sample toward the open end of the
capillary. The ablation plume may travel in a forward direction
congruent and/or parallel to the laser pulse. The capillary may
radially confine the ablation plume. The ablation plume may
comprise a collimated ablation plume. The ablation plume may
comprise a collinear ablation plume. The ablation plume may not be
hemispherical. The ablation plume may not be freely expanding. The
capillary may be oriented toward the electrospray plume. The
ablation plume may be ejected from the capillary toward the
electrospray plume. The ablation plume may be intercepted by an
electrospray plume and ionized to generate ions detectable by the
mass spectrometer.
[0063] In transmission geometry, the capillary dimensions, sample
volume, sample position in the capillary, position of the optical
fiber relative to the capillary and/or sample, and position of the
capillary relative to the electrospray apparatus and/or mass
spectrometer orifice may be optimized to improve ion production.
Referring to FIG. 8, the capillary may comprise an outer diameter
(OD), an inner diameter (ID), and a length (L). The capillary may
have an outer diameter (OD) from 7 mm to 100 .mu.m, such as, for
example 2 mm or 1 mm. The capillary may have an inner diameter (ID)
from 5 .mu.m to 5 mm, 10 .mu.m to 1000 .mu.m, 10 .mu.m to 30 .mu.m,
and 30 .mu.m to 50 .mu.m. The capillary may have a length (L) from
0.1 mm to 10 mm, 0.5 mm to 5 mm, and 0.1 mm to 1 mm. The capillary
may comprise a sample, such as, for example a single cell (C)
suspended in a liquid, such as an aqueous solution, having a sample
volume (V). The sample may have a sample volume from 1 picoliter to
100 .mu.L, 5 picoliters to 5 nL, 5 nL to 500 nL, 10 nL to 100 nL,
100 nL to 1 .mu.L, and 1 .mu.L to 100 .mu.L. In at least one
embodiment, the sample volume may be 100 nL to 100 .mu.L, 100 nL to
1 .mu.L, and 0.5 .mu.L. The sample may be positioned in a liquid
where the distance from the end of the capillary to the lower
meniscus of the liquid (d) may be from 0-10 mm, such as, for
example, 0 to 1 mm, greater than 0 to 1 mm, 0.25 mm, 0.5 mm, 0.75
mm, and 1 mm. Referring to FIG. 8, in various embodiments, the
focusing optics may comprise an optical fiber having a core
diameter (CD), a tip radius of curvature (R), a tip-to-liquid
distance (d-d.sub.i) and a tip insertion depth (d.sub.i). The
optical fiber core diameter (CD) may be from 15 .mu.m to 450 .mu.m,
such as, for example, 150 .mu.m, 250 .mu.m, 350 .mu.m, and 450
.mu.m. The tip radius of curvature (R) may be from 0.1 .mu.m to 25
.mu.m, such as, for example, 0.25 .mu.m to 5 .mu.m and 7.5 to 12.5
.mu.m. The tip insertion depth (d.sub.i) may be from 0 mm to 10 mm,
such as, for example, greater than 0 to 10 mm, 5 mm, 1 mm, and 0.5
mm. For example, the tip insertion depth (d.sub.i) may be 0 mm when
the tip is not inserted into the capillary. The tip-to-liquid
distance (d-d.sub.i) may be from 0 .mu.m to 50 .mu.m, such as, for
example, greater than 0 to 50 .mu.m, 1 .mu.m, 2 .mu.m, 5 .mu.m, 10
.mu.m, and 30 .mu.m. For example, the tip-to-liquid distance
(d-d.sub.i) may be 0 .mu.m when the tip contacts the lower meniscus
of the liquid. In various embodiments, the fiber tip may contact
the sample (d-d.sub.i=0 .mu.m). In at least one embodiment, the
tip-to-liquid distance (d-d.sub.i) may be twice the tip radius of
curvature (2R).
[0064] Referring to FIGS. 9A-C, in various embodiments, the device
may comprise one of a linearly tapered tip 50 and a curved tapered
tip 55. As shown in FIGS. 9A and 9B, the change in the radius of a
linearly tapered tip from the core diameter of the optical fiber to
the diameter of the tip is small relative to the change in the
radius of a curved tapered tip. The energy (illustrated in gray)
emitted from a curved tapered tip is illustrated in FIG. 9C. As
shown in FIG. 9C, a significant portion of the laser energy may be
emitted from the curved portion of the tip and/or the tip. Without
wishing to be bound to any particular theory, the linearly tapered
tip may exhibit less energy loss than the curved tapered tip. The
linearly tapered tip may provide more focused laser energy delivery
to the sample relative to a curved tapered tip. In various
embodiments, the tip may comprise a metal coating, such as, for
example, a silver coating, along the interior of the tip to reduce
energy loss. In various embodiments, the fiber may be heated and/or
drawn with a capillary puller to generate a linearly tapered tip
including a controlled taper angle. Without wishing to be bound to
any particular theory, a tapered tip may be characterized focusing
all or substantially all of its laser energy at the tip and/or
minimizing energy losses to provide more efficient sample ablation
relative to a conventional tip.
[0065] In various embodiments, the device may comprise a capillary
including a chemically modified interior surface. The chemically
modified surface may increase and/or decrease an interaction
between the capillary and sample. The capillary may comprise a
hydrophobic inner surface. The capillary may comprise a hydrophilic
inner surface. The capillary may be modified using hydrophobic
agents and/or hydrophilic agents, such as, for example, but not
limited to, pentafluorophenyldimethylchlorosilane, phenethylsilane,
trimethylsilane, hexamethyldisilazane,
3-aminopropyldimethylethoxysilane, and combinations thereof.
[0066] In various embodiments, the sample may comprise subcellular
components, a single cell, cells, small cell populations, cell
lines, and/or tissues. The single cell may have a smallest
dimension less than 100 micrometers, such as less than 50 .mu.m,
less than 25 .mu.m, and/or less than 10 .mu.m. The single cell may
have a smallest dimension from 1 .mu.m to 100 .mu.m, such as, for
example, from 5 .mu.m to 50 .mu.m, and 10 .mu.m to 25 .mu.m. In
various embodiments, the single cell may have a smallest dimension
from 1 .mu.m to 10 .mu.m. The small cell population may comprise 10
cells to 1 million cells, such as 50 cells to 100,000 cells, and
100 cells to 1,000 cells. The subcellular component may comprise
one or more of cytoplasm, a nucleus, a mitochondrion, a
chloroplast, a ribosome, an endoplasmic reticulum, a Golgi
apparatus, a lysosome, a proteasome, a peroxisome, a secretory
vesicle, a vacuole, and a microsome. In various embodiments, the
sample may comprise an aqueous droplet. In various embodiments, the
sample may comprise an aqueous droplet comprising subcellular
components, a single cell, cells, small cell populations, cell
lines, and/or tissues. In various embodiments, the sample may
comprise subcellular components, a single cell, cells, small cell
populations, cell lines, and/or tissues suspended in an aqueous
droplet. The sample may comprise a hydrophobic sample and/or a
hydrophilic sample. The sample may comprise one of a solid sample,
a liquid sample, and a solid suspended in an aqueous droplet.
[0067] In various embodiments, the sample may comprise water. For
example, tissue, cells and subcellular components may comprise
water. The sample may comprise a high, native water concentration.
The sample may comprise a native water concentration. In various
embodiments, the sample may comprise one of a cell and a small cell
population suspended in an aqueous solution. The aqueous solution
may comprise water, a buffer, such as, for example, HEPES or PBS,
cell culture media, such as, for example, RPMI 1640, BME, and Ham's
F-12, and/or any other suitable solution. The sample may comprise a
rehydrated sample. The sample may comprise a dehydrated sample
rehydrated with an aqueous solution. In various embodiments, the
rehydrated sample may be rehydrated via an environmental chamber
and/or an aqueous solution. The sample may comprise water and the
laser energy may be absorbed by the water in the sample. The sample
may be in a native environment and/or ambient environment.
[0068] In various embodiments, the capillary may be used to select
a sample for ablation and/or retrieve a sample for ablation. The
capillary may be used to capture the sample from a native
environment. As shown in FIG. 10A, a pulled glass capillary having
an inner diameter of about 100 .mu.m may be used to capture cells
by capillary action. As shown in FIG. 10B, the capillary extracted
about 15 cells. The capillary may use capillary forces to select a
sample for ablation and/or retrieve a sample for ablation. The
capillary may extract a liquid sample, a small cell population,
and/or a single cell, and/or a subcellular component into an
opening of the capillary via capillary forces. For example, the
capillary may extract untreated biological fluids, cells,
subcellular components, and tissue components from a sample in an
ambient environment for direct ablation. The extracted sample may
be positioned intermediate a first end of the capillary and a
second end of the capillary.
[0069] The capillary may have different inner diameters to
correspond to the sample volume. For example, the capillary may
have an inner diameter comparable to a single mammalian cell.
Without wishing to be bound to any particular theory, the inner
diameter of the capillary may affect the selection and/or retrieval
of the sample. For example, shearing forces may damage the cell
when the diameter of capillary entrance is smaller than the size of
the cell, and a capillary having a diameter greater than the size
of a single cell may extract more than one cell. A capillary having
a smaller inner diameter may exhibit improved plume collimation and
sampling relative to a capillary having a larger inner
diameter.
[0070] In various embodiments, the capillary may comprise a hollow
waveguide. A method for making Ag/AgI hollow glass waveguides is
described in U.S. Pat. No. 4,930,863, and Ag/AgI hollow glass
waveguides having bore diameters greater than or equal to about 300
.mu.m are commercially available from Polymicro Technologies, LLC.
As discussed above, the waveguide may couple the laser energy to
the sample, deliver the laser energy to the sample, collimate the
ablation plume, select a sample for ablation, and/or retrieve a
sample for ablation. The waveguide may be used to capture the
sample from a native environment. The waveguide may use capillary
forces to select a sample for ablation and/or retrieve a sample for
ablation. For example, the waveguide may extract untreated
biological fluids, cells, subcellular components, and tissue
components from a sample in an ambient environment for direct
ablation. The extracted sample may be positioned intermediate a
first end of the waveguide and a second end of the waveguide. The
waveguides may have different inner diameters to correspond to the
sample volume. The waveguide may have the same dimensions as the
capillary described above. For example, the waveguide may have an
inner diameter comparable to a single mammalian cell.
[0071] Referring to FIG. 11, in certain embodiments, a mass
spectrometer device may comprise a mid-infrared laser 1, such as,
for example, a Nd:YAG laser driven optical parametric oscillator, a
focusing system comprising a focusing device 21, such as, for
example, a lens and a beam steering device 22, such as, for
example, a mirror, a hollow waveguide held by a fiber mount 2, a
three dimensional translation stage having a sample mount 4, an
electrospray apparatus including an electrospray emitter 9, a
syringe pump 11, a high voltage power supply 10, a mass
spectrometer 12, and one or more long distance video microscopes 24
to visualize the sample when the sample is selected with the
waveguide and/or when the sample is positioned for ablation. The
waveguide may comprise the sample. The sample may be positioned
intermediate the first end of the waveguide and the second end of
the waveguide. The waveguide may deliver and/or couple the laser
energy to the sample. The ablation plume may be generated in the
waveguide. The ablation plume may travel in a forward direction
toward the second end of the waveguide. The waveguide may radially
confine the ablation plume. The ablation plume may comprise a
collimated ablation plume. The collimated ablation plume may
comprise a radially confined ablation plume. The collimated
ablation plume may comprise a collinear ablation plume. The
waveguide may be oriented toward the electrospray plume. The
ablation plume may be ejected from the waveguide toward the
electrospray plume.
[0072] In various embodiments, the mid-infrared laser pulse may
have a beam diameter of about 65% of the waveguide bore diameter.
The focusing lens may comprise a 50 mm focal length plano-convex
calcium fluoride lens. The long distance video microscope 24 may be
positioned orthogonal to the sample surface to visualize the
sampling by the hollow waveguide. The waveguide may be maneuvered
by a micromanipulator (not shown). The waveguide may contact a
sample comprising a single cell or cells to select and/or capture
the sample. The waveguide comprising the sample may be positioned
for sample ablation. The electrospray solution may comprise 50%
methanol solution and 0.1% acetic acid (v/v). Other electrospray
solutions and/or gas environments may be used to enhance ion
production and/or facilitate the fragmentation of the produced
ions. The syringe pump 11 may deliver the electrospray solution at
a rate of 300 nL/min. The high voltage power supply 10 may apply
about 3,100 V to the electrospray emitter 9 to generate a steady
electrospray plume. The distance and angle between the hollow
waveguide 23 and the electrospray axis may be adjusted to optimize
sampling conditions. In various embodiments, the distance between
the hollow waveguide 23 and the electrospray axis may be 1-15 mm,
such as, for example, 5 mm, 10 mm, or 12 mm, and the angle between
the hollow waveguide 23 and the electrospray axis may be
0-180.degree., such as, for example, 90.degree., 45.degree., and
5.degree..
[0073] In various embodiments, a method may comprise ablating a
sample by a laser pulse in a capillary to generate an ablation
plume, intercepting the ablation plume by an electrospray plume to
produce positive or negative ions, and detecting the ions by mass
spectrometry, wherein the ablation plume is a collimated ablation
plume. The collimated ablation plume may comprise a radially
confined ablation plume. The collimated ablation plume may comprise
a collinear ablation plume. In various embodiments, the capillary
may comprise a hollow waveguide. In various embodiments, the method
may comprise delivering the laser pulse to the sample by at least
one of focusing optics, an optical fiber, and a hollow waveguide.
The method may comprise coupling the laser pulse to the sample by
at least one of focusing optics, an optical fiber, and a hollow
waveguide. The laser pulse may comprise a mid-infrared laser
pulse.
[0074] In various embodiments, the method may comprise generating
an ablation plume in the capillary. The method may comprise
generating a radially confined ablation plume. The method may
comprise generating a collimated ablation plume. The method may
comprise generating a collinear ablation plume. The method may
comprise collimating the ablation plume with one of the capillary
and a hollow waveguide. As shown in FIG. 1B, a capillary may
collimate an ablation plume generated in the capillary. The
capillary may reduce and/or eliminate the radial expansion of the
ablation plume. The collimated ablation plume may improve ion
formation and/or ion efficiency. The method may comprise generating
an ablation plume in the hollow waveguide.
[0075] In various embodiments, the method may comprise ejecting at
least a portion of the ablation plume from the capillary. The
method may comprise ejecting at least a portion of the ablation
plume from the second end of the capillary. The ablation plume may
travel in a forward direction toward the second end of the
capillary. The method may comprise ejecting at least a portion of
the ablation plume from the second end of the capillary towards the
electrospray plume. The method may comprise ejecting a radially
confined ablation plume from the second end of the capillary. The
method may comprise ejecting a collimated ablation plume from the
second end of the capillary. The method may comprise ejecting a
collinear ablation plume from the second end of the capillary. The
method may comprise ejecting at least a portion of the ablation
plume from the hollow waveguide.
[0076] In various embodiments, the method may comprise subjecting
the sample to one of transmission geometry and reflection geometry
ablation. In reflection geometry, the method may comprise
delivering the laser pulse to a first side of the sample and
generating the ablation plume on the first side of the sample. In
transmission geometry, the method may comprise delivering the laser
pulse to a first side of the sample and generating the ablation
plume on a second side of the sample, such as, for example, an
opposite side of the sample. For example, the method may comprise
delivering the laser pulse to the rear of the sample and generating
an ablation plume on the front of the sample. In transmission
geometry, at least a portion of the ablation plume or at least a
substantial portion of the ablation plume may be on a side opposite
from the laser and at least a portion of the ablation plume or no
portion of the ablation plume may be on the same side as the laser.
In transmission geometry, the method may comprise ejecting at least
a portion of the ablation plume on a side of the sample opposite
from the laser.
[0077] In various embodiments, the method may comprise positioning
the sample intermediate a first end of the capillary and the second
end of the capillary. The method may comprise positioning the
sample proximate to the first end of the capillary. The method may
comprise positioning the sample adjacent to the first end of the
capillary. The method may comprise positioning the sample outside
the first end of the capillary. In various embodiments, the method
may comprise one or more of selecting and retrieving a sample for
ablation with the capillary. The method may comprise selecting
and/or retrieving the sample from a native environment with the
capillary using capillary forces. In various embodiments,
retrieving the sample may comprise capturing the sample from a
native environment with the capillary using capillary forces. As
shown in FIG. 10A, for example, a capillary may be inserted into an
aqueous droplet comprising cells to select one or more cells for
ablation. As shown in FIG. 10B, the cell or cells may be drawn into
the capillary by capillary forces.
[0078] Referring to FIGS. 5 and 6, in various embodiments, the
method may comprise hydrodynamically focusing the sample in a
stream of fluid. In various embodiments, a flow cytometer may
hydrodynamically focus the sample in a stream of fluid. In various
embodiments, a flow through capillary may hydrodynamically focus
the sample in a stream of fluid. The hydrodynamically focused
sample may comprise a single stream of cells. In various
embodiments, the method may comprise hydrodynamically focusing the
sample in a stream of fluid in a flow cytometer and/or a flow
through capillary, irradiating the stream of fluid with a
continuous laser on a first side of the capillary, detecting when
the sample passes the focused beam from the continuous laser, and
activating the mid-infrared laser when the sample is at a point of
ablation in the capillary. A cell may deflect the focused beam
emitted from the continuous laser 6. The detector may detect the
deflected laser beam and activate the delay generator 8. The delay
generator 8 may delay the activation of the mid-infrared laser 1
until the cell is at a point of ablation in the capillary. The
delay generator may trigger the mid-infrared laser pulse to ablate
the cell. The duration of the delay may be the time for the sample
to travel from the point when the cell intercepts the continuous
laser beam to the point of ablation proximate to or in the
capillary. In various embodiments, the method may comprise labeling
the sample with a fluorescent tag, such as, for example, green
fluorescent protein, yellow fluorescent protein, immunofluorescent
tag, or acridine orange dye. In various embodiments, the method may
comprise subjecting the sample to cell sorting through flow
cytometry prior to ablating the sample.
[0079] The various embodiments described herein may be better
understood when read in conjunction with the following
representative examples. The following examples are included for
purposes of illustration and not limitation.
[0080] An optical parametric oscillator (OPO) (Vibrant IR or
Opolette 100, Opotek, Carlsbad, Calif.) converted the output of a
100 Hz repetition rate Nd:YAG laser to mid-infrared laser pulses of
about 5 ns pulse length at about 2940 nm wavelength. Individual
laser pulses were selected using a high performance optical shutter
(SR470, Standford Research Systems, Inc., Sunnyvale, Calif.). In
certain embodiments, beam steering and focusing were accomplished
by gold coated mirrors (PF10-03-M01, Thorlabs, Newton, N.J.) and a
single 75 mm focal length plano-convex antireflection-coated ZnSe
lens or a 150 mm focal length plano-convex CaF.sub.2 lens (Infrared
Optical Products, Farmingdale, N.Y.). In certain embodiments, beam
steering and focusing were accomplished by a sharpened germanium
oxide (GeO.sub.2) optical fiber having a core diameter of 450 .mu.m
and a tip radius of curvature of 15 .mu.m to 50 .mu.m (HP Fiber,
Infrared Fiber Systems, Inc., Silver Spring, Md.). The optical
fiber was held in a bare fiber chuck (BFC300, Siskiyou Corp., Grant
Pass, Oreg.) that was attached to a five-axis translator (BFT-5,
Siskiyou Corporation, Grants Pass, Oreg.). The optical fiber was
positioned in contact with the sample. The optical fiber may
comprise a linearly tapered tip. In certain embodiments, beam
steering and focusing were accomplished by a hollow waveguide
having a 300 .mu.m bore diameter manufactured by Polymicro
Technologies, LLC. A 50 focal length plano-convex CaF.sub.2 lens
(Infrared Optical Products, Farmingtondale, N.Y.) was used to focus
the laser pulse onto the distal end of the optical fiber or hollow
waveguide.
[0081] The electrospray system comprised a low-noise syringe pump
(Physio 22, Harvard Apparatus, Holliston, Mass.) to feed a 50%
(v/v) aqueous methanol solution containing 0.1% (v/v) acetic acid
at 200-300 nL/min flow rate through a tapered stainless steel
emitter comprising a tapered tip having an outside diameter of 320
.mu.m and an inside diameter of 50 .mu.m. (MT320-50-5-5, New
Objective Inc., Woburn, Mass.). Stable high voltage was generated
by a regulated power supply (PS350, Stanford Research Systems,
Inc., Sunnyvale, Calif.). The regulated power supply provided 3,000
V directly to the emitter. The orifice of the mass spectrometer
sampling cone was on-axis with the electrospray emitter at a
distance of about 12 mm from its tip.
[0082] An orthogonal acceleration time-of-flight mass spectrometer
(Q-TOF Premier, Waters Co., Milford, Mass.) having a mass
resolution of 8,000 (FWHM) collected and analyzed the ions
generated by the LAESI source. No sample related ions were observed
when the laser was off. The electrospray solvent spectra were
subtracted from the LAESI spectra using the MassLynx 4.1 software
(Waters Co., Milford, Mass.).
[0083] To visualize the sample, a video microscope having a
7.times. precision zoom optic (Edmund Optics, Barrington, N.J.), a
2.times. infinity-corrected objective lens (M Plan Apo 2.times.,
Mitutoyo Co., Kanagawa, Japan), and a CCD camera (Marlin F131,
Allied Vision Technologies, Stadtroda, Germany) was positioned on
the capillary axis.
[0084] In certain embodiments, the ablation was performed in
transmission geometry. In transmission geometry, the optical fiber
was positioned inside the capillary from below and the ablation
plume was ejected from the opposite end. The capillary axis was 6.5
mm in front of the electrospray emitter tip. The capillary end that
ejected the ablation plume was 12 mm below the electrospray emitter
axis. The inner diameter of the capillary was 1 mm and the length
of the capillary was 3 mm.
[0085] Referring to FIG. 12, a representative mass spectrum in the
range of 0-1000 m/z was obtained from about twenty-five (25)
squamous epithelial cells. The squamous epithelial cells were
suspended in a 2.5 .mu.L droplet of water, positioned inside a
capillary having an inner diameter of 1 mm and a length of 3 mm,
and ablated by the mass spectrometric device in transmission
geometry. The inset in FIG. 12 includes an image of about
twenty-five (25) squamous epithelial cells stained with toluidine
blue. The scale bar in the inset is 50 micrometers. About
twenty-five (25) cells were selected from a large cell population
by diluting the cell population in water until its density was
sufficiently low such that the cells could be isolated and
retrieved from the solution with the capillary. The total cell
volume of a single cell, assuming a spherical shape, was about 10
picoliters to about 60 picoliters. The total cell volumes of the 25
cells, assuming a spherical shape, was about 25 times greater than
the total cell volume of the single cell.
[0086] FIGS. 13A-E include representative mass spectra in the range
of 0-600 m/z obtained from bradykinin dissolved in a 5 .mu.L
droplet of water. The samples were positioned inside capillaries
having an inner diameter of 2 mm and lengths of 2 mm, 3.8 mm, 5 mm,
6 mm, and 7.7 mm, respectively, and ablated by the mass
spectrometric device in transmission geometry. The optical fiber
was inserted into the droplet from the bottom of the capillary
prior to ablation. The total ion count for the representative mass
spectrum of bradykinin was 1610, 1230, 753, 690, and 481,
respectively. As shown in FIGS. 13A-E, the shorter capillaries
generally exhibited improved ionization efficiencies relative to
the longer capillaries. For example, the capillary having a length
of 2 mm had the highest total ion count, and thereby, the highest
ionization efficiency.
[0087] FIG. 14A includes a representative mass spectrum in the
range of 0-600 m/z obtained from 2.5 .mu.L of 0.1 mM bradykinin
solution in a capillary having an inner diameter of 1 mm and a
length of 2.5 mm. The sample was positioned inside the capillary
and ablated by the mass spectrometric device in transmission
geometry. The total ion count was 2460. FIG. 14B includes a
representative mass spectrum in the range of 0-600 m/z obtained
from 5 .mu.L of 0.1 mM bradykinin solution in a capillary having an
inner diameter of 2 mm and a length of 2.5 mm. The sample was
positioned inside the capillary and ablated by the mass
spectrometric device in transmission geometry. The total ion count
was 1610. As shown in FIGS. 14A and 14B, capillaries having smaller
inner diameters generally exhibited improved ionization
efficiencies relative to capillaries having larger inner diameters.
For example, the capillary having an inner diameter of 1 mm had the
highest total ion count, and thereby, the highest ionization
efficiency.
[0088] FIGS. 15A-D include representative mass spectra in the range
of 0-800 m/z obtained from squamous epithelial cells suspended in a
droplet of water. The samples were positioned inside a capillary
having an inner diameter of 1 mm and a length of 3 mm and ablated
by the mass spectrometric device in transmission geometry. FIG. 15A
includes a representative mass spectrum of 20 squamous epithelial
cells having a total ion count of 198. FIG. 15B includes a
representative mass spectrum of 10 squamous epithelial cells having
a total ion count of 91. FIG. 15C includes a representative mass
spectrum of 6 squamous epithelial cells having a total ion count of
50. FIG. 15D includes a representative mass spectrum of 4 squamous
epithelial cells having a total ion count of 34. As shown in FIG.
15D, a sample comprising 4 squamous epithelial cells exhibited
improved ionization efficiencies sufficient to generate ions
detectable by mass spectrometry. As shown in FIGS. 15A-D, the
signal intensity generally decreased as the number of cells
decreased.
[0089] FIG. 16 includes representative LAESI mass spectrum in the
range of 0-2000 m/z obtained from about less than 500 epithelial
beta cells having a size of about 5-10 .mu.m suspended in a 2.5
.mu.L droplet of water. The sample was positioned inside a
capillary having an inner diameter of 1 mm and a length of 2 8 mm
and ablated by the mass spectrometric device in transmission
geometry. The inset in FIG. 16 includes an image of a small cell
population of about 550 epithelial beta cells prior to
ablation.
[0090] In various embodiments, the dynamic range and/or limit of
detection may be improved relative to mass spectrometry systems
lacking a collimated ablation plume. FIG. 17 includes a graph
plotting signal intensity and concentration (molarity, M) for mass
spectrometry systems according to various embodiments described
herein and a mass spectrometry system lacking a collimated ablation
plume. Without wishing to be bound to any particular theory, a
collimated ablation plume may increase the dynamic range and/or
limit of detection relative to a mass spectrometry system lacking a
collimated ablation plume. As discussed above, mass spectrometry
system lacking a collimated ablation plume may comprise a freely
expanding ablation plume. A mass spectrometry system comprising a
freely expanding ablation plume may be characterized by lower
ionization efficiency, lower sensitivity, and/or lower limits of
detection because the ablation plume may freely expand in
three-dimensions and/or only a small portion of the ions is
captured by the electrospray plume. In various embodiments, the
capillary may reduce or eliminate the free radial expansion of the
ablation plume and/or generate a collimated ablation plume. Without
wishing to be bound to any particular theory, the collimated
expansion of the ablation plume may generate higher ionization
efficiency, higher sensitivity, and/or higher limits of detection
because a greater portion of the ions may be captured by the
electrospray plume. The collimated ablation plume may increase the
overlap of the ablation plume and electrospray plume. As shown in
FIG. 17, a mass spectrometry system according to various
embodiments described herein (.box-solid.) may comprise a dynamic
range of 6 orders of magnitude and a limit of detection of 600
attomoles. However, a mass spectrometry system lacking a collimated
ablation plume () may comprise a dynamic range of 4 orders of
magnitude and a limit of detection of 8 femtomoles. The inset in
FIG. 17 includes representative LAESI mass spectrum of 0.5 .mu.L of
1.2.times.10.sup.-9M verapamil solution comprising 50% (v/v) water
and 50% (v/v) methanol detected by a mass spectrometry system
comprising plume collimation.
[0091] All documents cited herein are incorporated herein by
reference, but only to the extent that the incorporated material
does not conflict with existing definitions, statements, or other
documents set forth herein. To the extent that any meaning or
definition of a term in this document conflicts with any meaning or
definition of the same term in a document incorporated by
reference, the meaning or definition assigned to that term in this
document shall govern. The citation of any document is not to be
construed as an admission that it is prior art with respect to this
application.
[0092] While particular embodiments of mass spectrometry have been
illustrated and described, it would be obvious to those skilled in
the art that various other changes and modifications can be made
without departing from the spirit and scope of the invention. Those
skilled in the art will recognize, or be able to ascertain using no
more than routine experimentation, numerous equivalents to the
specific apparatuses and methods described herein, including
alternatives, variants, additions, deletions, modifications and
substitutions. This application including the appended claims is
therefore intended to cover all such changes and modifications that
are within the scope of this application.
* * * * *